The present invention relates to Ras suppressors, in particular the Ras suppressor SUR-8.
The Ras family of proteins play critical roles in cell proliferation, differentiation, and cell migration in response to extracellular signals. Ras proteins are 21 kD membrane-bound GTPases that act as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. In the most well studied Ras-mediated signal transduction pathways, Ras is activated by receptor tyrosine kinases (RTK) through guanine nucleotide exchange factors that promote GTP binding and a change in Ras conformation to an active state (See e.g., McCormick, Nature 363, 15 [1993]). GTP-bound Ras then binds to the serine/threonine kinase Raf and recruits it to plasma membrane where it is activated. Once activated, Raf phosphorylates and activates the dual specific kinase MEK, which in turn phosphorylates and activates MAP kinase. Activated MAP kinase (MAPK) is proposed to regulate the activity of multiple targets including transcription factors for various physiological functions (Marshall, Curr. Opin. Genet. Dev. 4, 82 [1994]). Although this model for Ras-dependent signal transduction has been heavily studied, there has been almost no development or identification of effectors that regulate Ras signal transduction or that alter the associated cellular and physiological events stimulated by Ras. Little is known about the nature of Ras effectors or the pathways they control (Rubin et al., WO 97/21820 [1997]).
Recent studies using various model systems including biochemical studies in mammalian tissue culture and genetics in C. elegans and Drosophila suggest that the RTK-Ras-MAPK-mediated signal transduction pathway is not a simple linear pathway, but is likely part of complicated signal transduction network (Katz, and McCormick, Curr. Opin. Genet Dev. 7, 75-79 [1997]; Sundaram and Han, Cell 83, 889 [1995]; and Kornfeld, Trends Genet. 13, 55 [1997]). Thus, a series of converging and diverging signalling pathways are likely responsible for the diverse cellular responses mediated by Ras. In recent years, several potential Ras effectors in addition to Raf, including PI3 kinase and Ral GDS, have been described (Katz, supra) and are candidates for defining branch points of Ras signalling. However, these effectors cannot account for all of the cellular responses mediated by Ras (See e.g., White et al., Cell 80, 533 [1995]) and have not been sufficiently characterized.
Adding to the complexity of the various signaling processes is the collaboratory roles of multiple factors and signaling branches in regulating the output of the signal. The main players of the RTK-Ras-MAPK pathway may be essential elements of a given signaling process, but there are other factors that feed into or out of this pathway that may play important regulatory functions to ensure maximal activity of the pathway and to tighten the regulation of the signal. For example, the ksr genes were identified as suppressors of activated ras in C. elegans and Drosophila (Sundaram and Han, Cell 83, 889 [1995]; Kornfeld et al., Cell 83, 903 [1995]; and Therrien et al., Cell 83, 879 [1995]), however, their biochemical relation to the Ras pathway is still not well understood. In C. elegans, it has been shown that mutations in the ksr-1 gene do not obviously disrupt vulval signal transduction mediated by ras (i.e., a pathway controlled by ras in C. elegans). However, the ksr-1 activity becomes essential when the activity in the main pathway is compromised (Sundaram and Han, 1995, supra; and Kornfeld et al., 1995, supra).
The art is in need of additional regulators of the Ras signal transduction pathways. To gain regulatory control of Ras signalling and its physiological consequences (e.g., effects on cancer), new Ras effectors and their genes need to be identified and isolated. Without such developments, the ability to control Ras-mediated proliferation, differentiation, and cell migration will be severely limited.
The present invention relates to Ras suppressors, in particular the Ras suppressor SUR-8.
In one embodiment, the present invention provides an isolated nucleotide sequence encoding at least a portion of a SUR-8 protein. In some embodiments, the isolated nucleotide sequence encodes a SUR-8 protein selected from the group consisting of human SUR-8, murine SUR-8, and C. elegans SUR-8. In certain embodiments, the isolated nucleotide sequence is selected from the group consisting of SEQ ID NOS:1, 5, and 6. In another embodiment, the nucleotide sequence further comprises 5xe2x80x2 and 3xe2x80x2 flanking regions. In yet another embodiment, the nucleotide sequence further comprises intervening regions. In alternative embodiments, the nucleotide sequence comprises portions or fragments of the sequences described above.
In an another embodiment, the present invention provides vectors comprising a nucleotide sequence encoding at least a portion of SUR-8. In another embodiment, the present invention provides a host cell transformed with a vector comprising a nucleotide sequence encoding at least a portion of SUR-8. It is intended that the nucleotides, as well as the vector comprise deoxyribonucleotides and/or ribonucleotides. It is not intended that the vector be limited to any particular nucleotide sequences. It is also not intended that the host cell be limited to any particular cell type. The host cell may be contained within a living animal, as well as in culture (i.e., in cell cultures). In certain embodiments, the host cell is selected from the group consisting of bacteria, yeast, amphibian, and mammalian cells.
In one embodiment, the present invention provides an isolated peptide sequence comprising at least a fragment of SUR-8. In some embodiments, the isolated peptide sequence is selected from the group consisting of SEQ ID NOS:2, 7, and 8, and fragments thereof. The present invention also provides antibodies capable of specifically binding to any of the polypeptides described above. It is intended that the antibodies be produced using any suitable method known in the art, including polyclonal, as well as monoclonal antibodies.
The present invention also provides a polynucleotide sequence comprising at least fifteen nucleotides, capable of hybridizing under stringent conditions to at least a portion of an isolated nucleotide sequence encoding at least a portion of a SUR-8 protein. In certain embodiments, the polynucleotides sequence is selected from the group consisting of SEQ ID NOs:11-18.
The present invention also provides methods for detection of a polynucleotide encoding SUR-8 protein in a biological sample suspected of containing the polynucleotide encoding SUR-8, comprising the step of hybridizing at least a portion of the polynucleotide sequence capable of hybridizing under stringent conditions to at least a portion of an isolated nucleotide sequence encoding at least a portion of a SUR-8 protein, to nucleic acid of said biological sample to produce a hybridization complex. In one embodiment, the method further comprises the step of detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding SUR-8 in the biological sample. In some embodiments, the nucleic acid of the biological sample is amplified.
The present invention also provides a non-human animal overexpressing SUR-8 mRNA in the tissue of the non-human animal. In preferred embodiments, the SUR-8 is human SUR-8. However, SUR-8 mRNA from other species is also encompassed by the present invention. In one embodiment, the non-human animal is a member of the Order Rodentia.
The present invention also provides methods for producing the non-human transgenic animals, comprising the steps of introducing into an embryonal cell of a non-human animal a polynucleotide sequence encoding a SUR-8 protein; transplanting the embryonal target cell formed thereby, into a recipient female parent; and identifying at least one offspring containing the transgene, wherein the SUR-8 mRNA is overexpressed in the tissue of the offspring. In one embodiment, the SUR-8 mRNA is human SUR-8 mRNA. In an alternative embodiment, the SUR-8 protein is a mutant SUR-8 protein.
The present invention also provides a method for screening compounds for the ability to alter SUR-8 signal transduction (i.e., signal transduction pathways where SUR-8 is a component, either directly or indirectly), comprising providing: polypeptide sequence comprising at least a portion of SUR-8, polypeptide sequence comprising at least a portion of a protein known to interact (either directly of indirectly) with SUR-8; and one or more test compounds; combining in any order, the polypeptide sequence comprising at least a portion of SUR-8 and the polypeptide sequence comprising at least a portion of a protein known to interact with SUR-8, and the one or more test compounds; and detecting the presence or absence of an interaction (defined as any detectable interaction, such as covalent binding, physical association, direct or indirect activation or inhibition, etc.) between the polypeptide sequence comprising at least a portion of SUR-8 and the polypeptide sequence comprising at least a portion of a protein known to interact with SUR-8.